Epidermal Growth Factor (EGF) and its analogs represent a family of polypeptides having a variety of biological activities. Human EGF itself is a 53 amino acid polypeptide and its analogs vary in the number of amino acids in the polypeptide chain. A variety of these have been described in the literature. For example, U.S. Pat. No. 3,917,824 issued Nov. 4, 1975 EGF. The literature has also described various biological activities for these materials. Each material may or may not have the same activity or as broad a biological activity as the others but in general, it has been found that EGF and analogs inhibit the secretion of gastric acid and promote cell growth. Thus they have been useful in wound healing applications.
Human EGF is found in the urine of young males, in the maxillary glands, and in various other locations throughout the body. Present production techniques of human EGF and its analogs stem largely from isolation of the active components from urine due to a lesser extent, from the recombinant DNA method for preparing these materials. The difficulty inherent in the first of these is quite apparent. Isolation from urine sources is time consuming, expensive and relies on the supply of raw material. Furthermore, the isolation of intact human EGF is made difficult by the presence of closely related analogs. Current procedures leading to a recombinant method for producing EGF have not been entirely satisfactory because of apparent instability of human EGF that results during its production and purification. Some of the disadvantages will become more apparent as more detail is described in this specification.
EGF, also known as urogastrone, is known to contain 53 amino acids as shown in the following sequence: ##STR1##
The above formula is the formula for EGF as it exists in humans and as reported in the literature. The invention as described here, and for which more detail will be given later, relates to the microbial production of human EGF and some of its biologically active analogs. However, it is equally applicable to mouse EGF and in fact any EGF which has an equal or smaller number of glutamyl residues than human EGF.
It is to be noted in the sequence shown in formula I that residues 5, 24, 40 and 51 are glutamyl. The molecule in its natural form is folded in such a way that there are disulfide linkages between residues 6-20, 14-31, and 33-42.
While it is highly desirable to produce this material in recombinant DNA systems employing E. coli, there has been a significant obstacle to overcome because the E. coli tends to produce the EGF in its reduced form which is not stable to the bacterial endogenous proteases. It has been discovered and reported in the literature that in order to increase the stability, one should employ a leader sequence which results in an insoluble fusion protein and which therefore can be readily recovered from the cell paste. The selection of the specific leader sequence is known to be difficult and itself has created difficulty because at the end of the isolation phase of the polypeptide, the leader sequence must be separated and digested away from the EGF moiety at the N terminal amino group thereof. Similarly, even when an appropriate leader sequence is employed, great difficulty has been encountered in purifying the resulting polypeptide. It is often the case that chromatographic separations are required leading to a loss of product in an extremely tedious procedure.